The overall objective of this project is to elucidate the role of death domain-associated protein (Daxx) in regulating the tumor suppressor p53. p53 induces apoptosis, cell cycle, or senescence in response to a variety of stresses such as DNA damage and the activation of oncogenes. In unstressed cells, the potent anti-proliferative effects of p53 are restrained by Mdm2, a ubiquitin ligase (E3) that promotes rapid p53 ubiquitination and degradation. Stress signals activate p53 through the elevation of p53 levels, commonly via the disruption of the p53-Mdm2 interaction. In the case of DNA damage, this is due to phosphorylation of both p53 and Mdm2 and also due to Mdm2 self-ubiquitination. It is unclear how the cis- and trans-E3 activities of Mdm2, which have opposing effects on cell fate, are differentially regulated. The importance of p53 in tumor suppression is emphasized by the association of p53 mutations with nearly half of all human tumors. In the tumors that retain the wild type p53, p53 activity is often compromised through alterations in its regulatory proteins or effectors. However, for many tumors, the precise defects in the p53 pathway are not known. We recently found that Daxx plays a crucial role in the differential regulation of Mdm2's E3 activity. Daxx strongly promotes the stabilization of Mdm2 in unstressed cells by connecting Mdm2 to a de- ubiquitinase, Hausp. In addition, Daxx directly enhances the intrinsic E3 activity of Mdm2 towards p53. Upon DNA damage, Daxx dissociates from Mdm2, which precedes Mdm2 self-degradation and p53 activation. Furthermore, we found that Daxx is overexpressed in a large number of tumors. We hypothesize that Daxx is a crucial and multi-functional p53 inhibitor whose overexpression disables p53 in tumor cells. We will investigate the role of Daxx in modulating the E3 activity of Mdm2 (aim 1), elucidate the dynamic regulation of Daxx interactions and their contribution to p53 activation (aim 2), and determine the role of Daxx overexpression in tumorigenesis (aim 3). These studies will further define p53 regulation and its dysfunction in tumor cells. They may also reveal Daxx as a valuable diagnostic and therapeutic target in treating cancer patients.